In recent years, it has been shown that cancer is a genetic and epigenetic disease, where epigenetic and genetic alterations interact reciprocally to drive cancer development. However, unlike genetic mutations, epigenetic changes are reversible, and as such, drugs that restore the epigenetic balance represent exciting potential therapeutic targets for cancer. Epigenetics refers to the heritable changes in gene expression patterns that occur independently of alterations in primary DNA sequence. The main epigenetic mechanisms are DNA methylation and covalent histone modifications, which play important roles in the regulation of transcription.
G9a, also known as EHMT2, is a histone methyltransferase that mono- and dimethylates Lysine 9 of histone H3 (H3K9me1 and H3K9me2, respectively).
G9a expression is high in many cancers compared with normal tissue. Cancer transcriptome analysis has revealed high expression in many tumors including hepatocellular, colon, prostate, lung and invasive transitional cell carcinomas and in B cell chronic lymphocytic leukemia. In a number of human bladder and lung carcinoma patients, G9a expression is upregulated (Shankar S R. et al., Epigenetics, 2013. 8(1): p. 16-22). Knockdown of G9a in both bladder and lung cancer cell lines caused growth suppression and apoptosis. Studies on prostate cancer further corroborate its role in carcinogenesis, where downregulation of G9a causes centrosome disruption, chromosomal instability, inhibition of cell growth and increased cellular senescence in cancer cells. In aggressive lung cancer, high levels of G9a correlate with poor prognosis with increased cell migration and invasion in vitro and metastasis in vivo. G9a is also overexpressed in pancreatic adenocarcinoma and inhibition of G9a induces cellular senescence in this type of cancer. In Acute Myeloid Leukemia mouse models, loss of G9a significantly delays disease progression and reduces leukemia stem cells frequency.
DNA methylation is an epigenetic modification that modulates gene expression without altering the DNA base sequence and plays a crucial role in cancer by silencing tumor suppressor genes. DNA methyltransferases (DNMTs) are the enzymes that catalyze DNA methylation. DNMT1 encodes the maintenance methyltransferase and DNMT3A and DNMT3B encode de novo methyltransferase.
DNMT1 and DNMT3A/3B are overexpressed in several types of cancer such as breast, gastric, pancreas, prostate, hepatocellular, ovarian, renal, retinoblastoma, glioma or diffuse large B-cell lymphoma. Zebularine, decitabine and azacytidine inhibits cell proliferation and induce apoptosis in acute lymphoblastic leukemia, acute myeloid leukemia, hepatic carcinoma, lung, breast, gastric or cervical cancer among others (Vilas-Zornoza A. et al., PLoS ONE, 2011. 6(2): p. e17012). Decitabine has been currently approved for myelodysplastic syndrome by the US Food and Drug Administration.
However, many efforts are made to develop new non-nucleoside inhibitors to overcome the limits of these azanucleosides, such as chemical instability and incorporation into DNA for activity.
A series of quinazoline derivatives have been described as potent selective G9a/GLP inhibitors, such as N-(1-benzyl-4-piperidyl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (also known as BIX01294), 2-cyclohexyl-N-(1-isopropyl-4-piperidyl)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazolin-4-amine (also known as UNC0638), and 2-(4,4-difluoro-1-piperidyl)-N-(1-isopropyl-4-piperidyl)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazolin-4-amine (also known as UNC0642). However, these molecules display activity against DNMTs at high micromolar IC50 values at most. Thus, for compound BIX01294 a 35±8% inhibition against DNMT1 and 12±3% inhibition against DNMT3A at 100 μM has been reported, which would correspond to IC50 values>100 μM against DNMT1 and DNMT3A, respectively (Rotili D. et al., PLoS ONE, 2014. 9(5): p. E96941). For compound UNC0638 an IC50 value of 107±6 μM against DNMT1 has been reported (Vedadi M. et al., Nat. Chem. Biol. 2011, 7, pp. 566-574), whereas for compound UNC0642, an in vitro IC50 value>50 μM against DNMT1 has been described (Liu F. et al., J. Med. Chem. 2013, 56(21), pp. 8931-42).
Cellular reprogramming is a process that includes the induction of pluripotency in differentiated cells, generating induced pluripotent stem cells (iPSC), and the direct conversion of those differentiated cells to a non-related cell type, process called direct reprogramming. The generation of iPSC produces cells with similar but not identical properties to natural pluripotent stem cells, ie, embryonic stem cells (ESC). In general iPSC have been described to be similar to ESC in morphology, proliferation, teratoma formation and differentiation efficiency, but remarkable epigenetic and gene expression differences have been also observed. However, generation of iPSC may relay some knowledge about innate genetic aspects that occur during natural embryonic development.
Since their discovery, it was clear that cellular reprogramming, and especially iPSC generation, were destined to revolutionize the field of medicine. The power to create patient-specific pluripotent cells promised to provide invaluable models of human disease for in vitro research and offered the prospect of autologous, rejection-proof cell transplantation therapies and new regenerative medicine approaches Reprogramming methods that utilize viral vectors were however judged too risky to be used in clinical therapies. Thus, most efforts on the field have been focused on development of different approaches to generate good quality and safer transgene-free or integration-free iPS cells. This is an area of research where chemical biology has made a significant contribution to facilitate the efficient production of high quality iPSCs and elucidate the biological mechanisms governing their phenotype. In particular the development of various small molecules (Jung D W., et al. ACS Chem. Biol, 2014. 9(1): p. 80-95) has achieved a pivotal role in optimizing protocols for iPSC production identifying small-molecule combinations that were able to drive the reprogramming of mouse somatic cells toward pluripotent cells.
Moreover, it has been described that some epigenetic marks, like DNA and H3K9 methylation, may have an important role in cell reprogramming.
The reprogramming efficiency of BIX01294 obtained by Oct-4 and Klf-4 overexpression in mouse embryonic fibroblasts has been reported in Shi Y. et al., Cell Stem Cell 2008, 3, pp. 568-574. Besides, this paper also discloses that (2S)-2-(1,3-dioxoisoindolin-2-yl)-3-(1H-indol-3-yl)propanoic acid (also known as RG108), a DNMT inhibitor, enhanced the reprogramming activity in the presence of BIX01294.
There is still a need of developing compounds which show improved activity in the treatment and/or prevention of cancer and in the generation of induced pluripotent stem cells.